The present inventions involves the large-scale production of membrane specific proteins and more particularly involves the production of such proteins using transgenic animals and most specifically involves the production of CFTR, the protein involved with Cystic Fibrosis.
Striking progress has been made in our understanding of cystic fibrosis (CF) during the period since the gene associated with the disease was identified1-3. Many mutations within the gene have been identified in DNA from patients with CF4-10. The protein product of the gene, named CFTR, has been identified11 and functional studies have shown that CFTR cDNA is able to complement the defect in ion transport characteristic of cells from CF patients12,13. The domain structure of CFTR has been probed by analysis of mutated versions of the protein14-18. Such studies indicate that CFTR is a Clxe2x88x92channel14,19,20 and that it is regulated by phosphorylation of the R-domain16,18,21 and by nucleotide binding22. To our knowledge none of such proteins have involved exogenous or recombinant membrane proteins.
In accordance with the principles of the present invention it has been surprisingly discovered that exogenous or recombinant membrane proteins can be produced in the milk of transgenic animals. In the most preferred embodiment of the present invention, recombinant CFTR has been produced in the milk of transgenic mice containing CFTR cDNA downstream of a mammary specific promoter. Most preferred embodiments will employ larger transgenic animals including, for example, rabbits, goats, sheep and cows which produce large quantities of milk however, the development of such animals takes considerably longer than the time periods required for developing transgenic mice. While the time periods vary considerably, the procedures and methods are substantially identical.
Other embodiments include the apocrine like secretion in tissue culture of membrane proteins by cells which have been maintained in a differentiated state. Such proteins would be secreted as part of the membrane released during the xe2x80x9cpinching offxe2x80x9d proces. Membrane proteins suitable for production according to the methods of the present include receptors, channels, viral glycoproteins, transporters and other proteins typically associated with cellular membranes. Such membrane proteins can be used for therapeutics such as by administration of the abnormally deficient or missing protein, or by use as vaccines in the case of viral glycoproteins including for example utilization of the envelope of HIV, Herpes Virus or influenza in order to develop immunity with respect to infection caused thereby.
Advantageously, methods of the present invention can also be employed for the production of membrane proteins useful for diagnostic purposes. For example, one could use the present methods to produce the receptor for thyroid stimulating hormone (TSHR) which would be useful for the diagnosis of Graves Disease. Such proteins could also be used to screen for therapeutically active compounds.
Preferred methods of the present invention will employ promoters, ideally coupled with suitable enhancers, that are mammary specific so that production of the desired membrane protein is incorporated into milk fat globules which will appear in the milk of a lactating female transgenic animal. Such an animal is, of course, by definition, reproductively competant. Greater appreciation of these and other emodiments will be acquired by study of the following drawings, examples and detailed procedures.